Draft genome sequence and probiotic functional property analysis of Lactobacillus gasseri LM1065 for food industry applications

Probiotics are defined as live organisms in the host that contribute to health benefits. Lactobacillus gasseri LM1065, isolated from human breast milk, was investigated for its probiotic properties based on its genome. Draft genome map and de novo assembly were performed using the PacBio RS II system and hierarchical genome assembly process (HGAP). Probiotic properties were determined by the resistance to gastric conditions, adherence ability, enzyme production, safety assessment and mobile genetic elements. The fungistatic effect and inhibition of hyphae transition were studied using the cell-free supernatant (CFS). L. gasseri LM1065 showed high gastric pepsin tolerance and mild tolerance to bile salts. Auto-aggregation and hydrophobicity were measured to be 61.21% and 61.55%, respectively. The adherence to the human intestinal epithelial cells was measured to be 2.02%. Antibiotic-resistance genes and putative virulence genes were not predicted in the genomic analysis, and antibiotic susceptibility was satisfied by the criteria of the European Food Safety Authority. CFS showed a fungistatic effect and suppressed the tricarboxylic acid cycle in Candida albicans (29.02%). CFS also inhibited the transition to true hyphae and damaged the blastoconidia. This study demonstrates the essential properties of this novel probiotic, L. gasseri LM1065, and potential to inhibit vaginal C. albicans infection.

Mobile genetic elements and genomic island. Mobile genetic elements (MGEs) in L. gasseri LM1065 were shown in Table S3. Total 7 of prophages, 2 of integrative and conjugative elements (ICEs), 57 of transposon insertion-sequence (IS) or IS cluster were detected in L. gasseri LM1065. In genomic island (GI) analysis, pathogenicity island and antibiotics resistance island were not found in L. gasseri LM1065. Plasmid sequences were not detected in L. gasseri LM1065. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) which protect against MGEs were detected in L. gasseri LM1065.

Discussion
Probiotics are live microorganisms that contribute to host health when adequate amounts are consumed 2,15-17 .
Probiotics are required to survive through the GIT and colonize the small intestine and colon for a long period 6 with a safety guarantee 18,19 . L. gasseri LM1065 exhibited survival under gastric conditions, enzyme production, colonization and adhesion ability without antibiotic resistance, hemolysis, and putative virulence factors compared to functional gene annotation. Probiotics are exposed to various types of stress during freeze-drying 20 , containing a food matrix and passing through the GIT 16 . These external stresses can damage cell membranes, induce the release of internal beneficial enzymes, and inhibit the colonization of the intestine 6,20 . In the present study, stress-responsive functional genes, including acid shock proteins, were investigated in L. gasseri LM1065 (Table 1). L. gasseri LM1065 showed high acid and mild bile salt tolerance ( Table 2). Tang et al. 21 reported that the bile salt concentrations vary from 0.03 to 0.3% in the small intestine during food digestion. L. gasseri LM1065 can survive in low concentrations of bile salts within the small intestine. Li et al. 17 suggested that mild bile salt tolerance results from the microenvironment of the microbial niche. Lactobacillus taiwanensis, isolated from Peyer's patches, also showed mild tolerance to bile salt because Peyer's patches show mild bile salt conditions compared to other small intestinal sites. These properties were also observed in Limosilactobacillus fermentum 4LB16 and 10LB1 isolated from the human vaginal tract 22 .
In general, bacterial adhesion to the human intestinal tract is mediated by cell surface components. In Lactobacillus, surface-layer proteins, cell wall-anchored mucus-binding proteins, cell-surface collagen-binding proteins, and mannose-specific adhesins have been reported to adhere to host molecules and mechanisms 23 . The cell wall-anchored protein (CWAP) contains five amino acid motifs, LPXTG, which is constructed from Leu-Pro-any amino acid-Thr-Gly 24 . Gram-positive bacteria utilize sortase to cleave surface proteins, particularly the Thr-Gly residue of LPXTG. The cleaved threonine residue mediates cell wall attachment 25 . Zhang et al. 23 studied the transformation of LPXTG into Lactococcus lactis, which confers adhesion to human epithelial cells. L. gasseri LM1065 showed higher auto-aggregation and adherence than L. rhamnosus ATCC 53103 (Table 2). Table 2. Acid tolerance, bile salt tolerance, auto-aggregation, hydrophobicity and adherence to intestinal epithelial cell ability of Lactobacillus gasseri LM1065. Data are shown as means ± standard deviations of three independent experiments. Significant difference is compared to Lacticaseibacillus rhamnosus ATCC 53103 (P < 0.01).   www.nature.com/scientificreports/ LPXTG and sortase were predicted in L. gasseri LM1065 (Table 1), and these genetic properties contribute to its essential probiotic properties. Gut-microbiomes produce enzymes that behave in complementary ways in human metabolism 26 . In the gut microbiome niche, LAB play critical roles in digestion, nutrient absorption, and improvement of nutritional value using numerous enzymes 27 . Most represent enzymes in Lactobacillus are lactase, β-galactosidase, glycosidase, protease, lipase, esterase and phytase 26,27 . Otherwise β-glucuronidase, which is released by Escherichia, Clostridium, and Staphylococcus 26 , is associated with potential carcinogenic metabolite conversion 19,26 . L. gasseri LM1065 released 11 enzymes, including β-galactosidase whereas β-glucuronidase activity was not found (Table S2). β-Galactosidase is an important biotechnological source in the food industry. β-Galactosidase is applied to ice cream to prevent undesirable crystallization and improve its creaminess. In addition, β-galactosidase is used in bakeries to improve sweetness 27 . Lactose intolerance occurred in a person lacking β-galactosidase in the  www.nature.com/scientificreports/ intestine and caused a lack of β-galactosidase accumulation of lactose. Excessive lactose affects osmotic pressure, resulting in digestive disorders 26 . Thus, β-galactosidase produced by gut-microbes attenuate digestive disorders in lactose intolerance consumers 26,27 . Interestingly, L. gasseri LM1065 was predicted to contain β-galactosidase and glycoside hydrolase family 1 involved in lactose hydrolysis (Table 1).
In the host immune system, pattern recognition receptors (PRRs) play a critical role in recognizing pathogens from the external environment via pathogen-associated molecular patterns (PAMPs) 28,29 . The cell wall of C. albicans consists of two parts: mannosylated protein (outer layer) and β-1,3-glucan with underlying chitin (inner layer). Dectin-1 is a major PRR that recognizes β-1,3-glucan as a PAMP 29 . β-1,3-glucan is not only a PAMP but also a biofilm component that resists stress 30 . Therefore, β-1,3-glucan regulation is considered an effective strategy for the treatment of C. albicans and VVC 30,31 . L. gasseri LM1065 was predicted to contain the β-glucanase gene (Table 1), which inhibits the growth and hyphae transition of C. albicans ATCC 11006 (Fig. 4). Additionally, L. gasseri LM1065 was predicted to produce gassericin T (Table 1 and Fig. 1E).

Conclusions
L. gasseri LM1065, isolated from human breast milk, has essential probiotic properties, including resistance to gastric conditions and adherence to intestinal cells. L. gasseri LM1065 satisfied safety requirements, including antibiotic resistance and putative virulence factor genes. Additionally, L. gasseri LM1065 was suggested as a probiotic for women, as the bacteria can inhibit candidiasis by suppressing the TCA cycle in C. albicans and blocking its transition to hyphae.

Materials and methods
Subjects and isolation of Lactobacillus gasseri LM1065. The collection of human breast milk were approved by Institutional Review Board of the Lactomason according to Enforcement Decree of Bioethics and Safety Act in Korea. All donors provided written informed consent before enrollment in the study and all methods were carried out in accordance with the Declaration of Helsinki. Three human breast milk samples were donated by three healthy women (25-40 years old) living in Gyeongsangnam-do on May 24, 2017. The participants did not have any underlying conditions and took any antibiotics or probiotics prior to the study for at least three months 18 . Human breast milk specimens were placed in a sterile container and diluted 10 times with phosphate-buffered saline (PBS, pH 7.2). The diluted specimens were spread on de Man-Rogosa-Sharpe (MRS) agar (for Lactobacillus) (BD Biosciences, Franklin Lakes, NJ, USA), M17 agar (for lactic Streptococcus and Lactococcus) (MBcell, Seoul, South Korea), and Bifidobacterium selective agar (for Bifidobacterium spp.) (MBcell). The spread agar plates were then incubated at 37 °C for 48 h. After 48 h, colonies isolated from MRS agar were spread on bromocresol purple (BCP) agar, and yellow colonies on BCP agar were further purified in newly prepared MRS agar until a single colony was obtained. Single and pure colonies were enriched in MRS broth for Gram staining and catalase reactions. The isolate was identified as a Gram-positive catalase-negative rod-type strain. The isolated strain was named LM1065 based on the institution's naming system and identified by 16S rRNA sequencing as L. gasseri. L. gasseri LM1065 was stored in MRS containing 20% glycerol at − 80 °C until use 32 . Microorganisms and conditions. L. rhamnosus ATCC 53103 and C. albicans ATCC 11006 were purchased from the American Type Culture Collection (ATCC). L. gasseri LM1065 and L. rhamnosus ATCC 53103 were cultured in MRS broth at 37 °C with aerobic condition and sub-cultured three times every 12 h until use. C. albicans ATCC 11006 was cultured in yeast mold (YM) broth at 24 °C for 48 h with aerobic condition until use.
Extraction of genomic DNA and genome analysis. L. gasseri LM1065 was harvested by centrifugation at 12,000 rpm at 4 °C for 10 min. The harvested bacterial cells were washed with PBS and genomic DNA (gDNA) was extracted. gDNA was extracted from L. gasseri LM1065 using the TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit (Takara Bio, Kusatsu, Japan) according to the manufacturer's guidelines.
A total of 5 μg of the gDNA sample was used for library preparation. A DNA library was constructed and sequenced using single molecular real-time (SMRT) sequencing technology (Pacific Biosciences, Menlo Park, CA, USA). The SMRTbell library preparation was performed with the SMRTbell Template Prep Kit 1.0, and DNA/Polymerase Biding kit P6. The SMRT library was sequenced using 1 SMRT cell using C4 chemistry (DNA sequencing Reagent 4.0) and 240-min movies were captured for each SMRT cell using the PacBio RS II system by Insilicogen (Yongin, South Korea). The genome coverage (depth of coverage) was 195 × and HiFi long-read sequencing perform for analysis.
De novo assembly was performed in the hierarchical genome assembly process (HGAP) including consensus polishing workflow using Quiver, and 1,929,825 bp of N50 contig and 2,251,884 bp of total contig were obtained. Sequence alignment and contig formation were performed using MUMmer 3.5. The coding sequence was predicted using GLIMMER 3.0, and the GO analysis was performed using Blast2GO 33 . The plasmid was predicted using nucleotide BLAST and bacteriocin gene clusters were predicted and visualized by antiSMASH bacterial version 7.0.0 34 .
Multiple alignment and genomic comparison were analyzed using MAUVE 35 .
Phylogenetic analysis based on orthologous gene and comparing of average nucleotide identity. Phylogenetic relationships of L. gasseri LM1065 were constructed based on ortholog gene sequences.
The whole genome sequences of Lactobacillus and Lactiplantibacillus were obtained from the National Center for Biotechnology Information (NCBI) database. Ortholog analysis was performed using OrthoFinder v2.5.4 and species tree was inferred using STAG algorithm and rooted using STRIDE algorithm in OrthoFinder 36 . The species tree was illustrated by Dendroscope 3 37 . www.nature.com/scientificreports/ The ANI values were estimated using the OrthoANI algorithm in EzBioCloud (https:// www. ezbio cloud. net/ tools/ ani) 38 . The result of ANI distance was generated using the heatmap plot function of the TBtools 39 .
Cellular fatty acid analysis of Lactobacillus gasseri LM1065. Cellular fatty acid extraction of L. gasseri LM1065 was performed using the Bligh and Dyer method with modifications 20 . In brief, 200 μL of chloroform/methanol solution (2:1, v/v) and 300 μL of 0.6 M hydrochloric acid solution (in methanol) were added to 20 mg of lyophilized L. gasseri LM1065. The mixture was shaken vigorously for 2 min and then incubated at 85 °C for 60 min. The reaction mixture was cooled at 25 °C for 20 min, and fatty acid methyl esters (FAME) were extracted using n-hexane for 60-120 min. The FAME extracted layer (n-hexane layer) was transferred into a clear vial and stored at − 20 °C until analysis.
Cellular fatty acid analysis was performed using Gas Chromatography/Mass selective detector (GC/MSD). The GC/MSD system was composed of an Agilent 8890 gas chromatography system coupled with a 5977 B mass selective detector (MSD) and a 7693A automated liquid sampler (Agilent, Santa Clara, CA, USA). An Agilent J&W DB-FastFAME capillary column packed with cyanopropyl (30 m × 0.25 mm, 0.25 μm) was employed. The injection port temperature was 250 °C under constant flow, and 1 μL of the sample was injected using the split mode of 20:1. Ultrapure helium was used as the carrier gas at a flow rate of 1 mL/min. The initial oven temperature was 60 °C for 1 min, raised from 60 to 165 °C at a rate of 60 °C/min, held for 1 min at 165 °C, raised from 165 to 230 °C at a rate of 5 °C/min, and maintained for 3 min. The temperatures of the ion source and transfer line were 230 °C and 250 °C, respectively. Mass spectra were obtained using electron ionization (EI) at 70 eV and recorded m/z 40-550 of mass range. Methyl undecanoate was used as an internal standard 40 . Tolerance to pepsin and bile salt. The resistance properties of L. gassseri LM1065 to artificial gastric conditions were investigated in a previous study with modifications 2,[15][16][17]21,22 . L. gasseri LM1065 was inoculated into MRS containing 0.3% pepsin (pH 2.5) or oxgall (0.05, 0.1, 0.2, and 0.3%) and incubated at 37 °C. After 2 and 24 h of incubation, viable cells in pepsin-and oxgall-containing MRS broths were measured by spreading on MRS agar. L. rhamnosus ATCC 53103 was used as the control.
Auto-aggregation and cell surface hydrophobicity. L. gasseri LM 1065 was harvested by centrifugation at 12,000 rpm and 4 °C for 10 min and washed twice with PBS. The washed cells were resuspended in PBS and adjusted to an OD 600 of 0.5.
To evaluate the auto-aggregation of L. gasseri LM1065, the adjusted bacterial suspension was allowed to stand and incubated at 37 °C. The upper suspension was collected (4 and 24 h), and the absorbance was measured at 600 nm. Auto-aggregation was calculated using the following equation: where A 0 is the initial absorbance (0.5), and A time is the absorbance of the supernatant at 4 and 24 h, respectively 2,11 . L. rhamnosus ATCC 53103 was used as the control.
To evaluate the hydrophobicity of L. gasseri LM1065, 2 mL of the adjusted bacterial suspension was mixed with 1 mL hexadecane. The mixture was then allowed to stand at 25 °C for 30 min. After incubation, the aqueous phase was separated, and its absorbance was measured at 600 nm. The hydrophobicity was calculated using the following equation.
where A 0 is the initial absorbance (0.5) and A 30 is the absorbance of the aqueous phase at 30 min 18,40 . L. rhamnosus ATCC 53103 was used as the control. penicillin-streptomycin solution at 37 °C in a humidified atmosphere containing 5% CO 2 . During incubation, the media were changed every 2-3 days, and cells were grown to 80% confluence. When HT-29 cells reached 80% confluence, the adherent cells were trypsinized with trypsin-EDTA solution (0.25%) and harvested by centrifugation. Harvested cells were seeded in 24-well plates (1 × 10 5 cells/well) and incubated with changing media to form a monolayer. Activated L. gasseri LM1065 was diluted to approximately 8 Log CFU/mL, and the HT-29 monolayer was treated with diluted L. gasseri LM1065 for 2 h without antibiotics. After 2 h, non-adherent bacterial cells were washed using PBS, and adherent bacterial cells were collected using 1% (v/v) Triton-X solution. Adherent bacterial cells were spread on MRS agar and viable cells were estimated. L. rhamnosus ATCC 53103 was used as the control.
Hemolysis activity was determined using Columbia blood agar (Oxoid, Basingstoke, United Kingdom) containing 5% sheep blood.
Minimum inhibitory concentration and fungistatic effect of Lactobacillus gasseri LM1065. The cell-free supernatant (CFS) of L. gasseri LM1065 was prepared using a 0.45 μm cellulose acetate membrane filter. To determine the minimum inhibitory concentration (MIC), the CFS was diluted in a 96-well plate using YM broth (MBcell, Seoul, South Korea). After dilution, approximately 6 Log CFU/mL of C. albicans ATCC 11006 was added to each well and further incubated at 24 °C for 48 h. MIC was determined as the lowest concentration that did not show C. albicans growth visually 45 . To measure the fungistatic effect of CFS, approximately 7 Log CFU/mL of C. albicans ATCC 11006 was treated with different CFS concentrations (0.5, 1.0, and 1.5 × MIC) and incubated at 24 °C for 48 h. After incubation, C. albicans was spread on YM agar, further incubated at 24 °C, and viable cells were counted.
Tricarboxylic acid cycle inhibition and microscopic observation. Tricarboxylic acid (TCA) cycle activity in CFStreated C. albicans ATCC 11006 was measured using an iodonitrotetrazolium chloride (INT; Sigma-Aldrich, MO, USA) solution 44 . Briefly, approximately 7 Log CFU/mL of C. albicans ATCC 11006 was treated with 0.5, 1.0 and 1.5 × MIC of CFS and incubated for 24 h. After incubation, C. albicans was collected by centrifugation at 3,000 rpm at 4 °C for 10 min and washed twice with PBS. The harvested C. albicans was diluted to an OD 600 of 0.1, and INT solution (1 mM final concentration) was added. The INT solution-treated C. albicans cells were incubated at 37 °C for 30 min. The TCA cycle was assessed by measuring the absorbance of formazan at 630 nm.
For microscopic observation, the harvested cells were stained with 0.4% trypan blue solution (Gibco). The morphology of the CFS-treated C. albicans was observed using a BX53 biological microscope (Olympus, Tokyo, Japan). All observations were performed at a total magnification of 400 × . Microscopic images were obtained using the eXcope software. Morphological information was obtained from a previous study 46 .
Statistical analysis. Statistical analyses were performed using the SPSS Statistics version 18 software (IBM, Armonk, NY, USA). Mean values were analyzed using the t-test and one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests and Tukey's range test at P < 0.05.
Ethical consideration. The collection of human breast milk were approved by Institutional Review Board of the Lactomason according to Enforcement Decree of Bioethics and Safety Act in Korea. All donors signed an informed consent form before enrollment in the study and voluntarily provided samples for only research purpose in accordance with the Declaration of Helsinki.

Data availability
The draft genome sequence has been deposited at DDBJ/ENA/GenBank under the accession JAQOUF000000000 (https:// www. ncbi. nlm. nih. gov/ nucco re/ JAQOU F0000 00000). The authors confirm that the data of this study are available within the article. www.nature.com/scientificreports/